Adhesive protein and uses thereof

ABSTRACT

An adhesive protein, present inter alia in the stickleback nest building glue has now been isolated and purified. Its amino acid sequence and corresponding coding polynucleotide sequences are isolated and sequenced. The protein finds both medical and technical uses, whereas both the protein and the polynucleotide sequence can be used in analyses, determining the presence and influence of androgenic substances, for example androgenic pollutants, present in the aquatic environment.

FIELD OF THE INVENTION

[0001] This invention concerns an adhesive protein, isolated andpurified by the present inventors, and its uses in analytical, medicaland technical applications. Further, the nucleotide sequence encodingsaid protein and its amino acid sequence are disclosed, including usesthereof.

BACKGROUND OF THE INVENTION

[0002] Analytical Applications:

[0003] The increasing pollution of the environment is one consequence ofour highly developed technical society. It has quite recently beendiscovered, that certain organic compounds, both pharmaceuticals andindustrial chemicals, such as softening additives in plastics, arehormonally active or hormone mimicking in living organisms.Unmetabolized etinyle oestradiol from oral contraceptives has beendetected in municipal effluents. Pulp mills are another possible sourceof hormonally active compounds, such as phytoestrogens. There are nowalarming indications that these compounds interfere with both earlyembryo and larval development as well as sexual differentiation in waterliving animals. Besides estrogenic responses, masculinization of femalefish has been observed.

[0004] Present models for detecting these compounds and monitoring theireffects include observations (biopsies) of fish and frogs in the areaunder investigation, experiments involving laboratory animals etc.Hitherto, cDNA probes have been developed for the oestrogen receptor(ER), vitellogenin (VTG) and vitelline envelope proteins (VEP).

[0005] There remains however an urgent need for the development of new,more sensitive markers for hormonal responses such as androgenicresponses.

[0006] Medical Applications:

[0007] Bio-degradable gels, adhesives and, in some applications, bindersand fillers, are needed in modern health care. Wound healing isdisturbed by the presence of necrotic tissue. Severe burns, severeulcers, deep cuts etc where necrotic tissue is present and difficult toremove, are today treated with waterbased gels, softening and removingnecrotic tissue. One gel, presently on the market contains water, sodiumcarboxymethyl cellulose, and calcium alginate.

[0008] Alginates are hydrocolloids obtained from seaweed. They are welltolerated by the body and biologically degradable. Alginates are used inhealth care as components in wound dressings, fillers for deep and/orexudating wounds, compresses etc. Alginate products are usuallydelivered in the forms of pads, ropes or ribbons. When absorbing theexudate, the alginate products form a gel which maintains a moistenvironment and protects the wound.

[0009] Another biologic polymer used in health care is collagen, whichplays an important part in wound healing. Collagen can be extracted fromanimal sources and used as a matrix for wound healing, skinreconstruction etc. Artificial skin grafts, used in dermatoplasticsurgery and reconstructive surgery, are presently based on animal skin,which has been subjected to chemical treatments, leaving only thesupporting collagen matrix structure, which will be colonised by thepatients own tissue.

[0010] Biodegradable adhesives are used for closing of minor wounds andincisions, for example in facial wounds and plastic surgery. Likewise,biodegradable sutures are currently in use.

[0011] Technical Applications:

[0012] The development of a glue which would be both possible to applyand to cure in moist environments or even under water, is highlydesired. Presently used glues and sealing agents are either epoxy based,cement based or based on synthetic polymers. Both the epoxy compoundsand the synthetic polymers may leach and constitute a risk to theenvironment. Their application often require mechanical working orkneading of the glue or sealing agent, in order to remove the waterpresent on the surfaces. There is a need for new glues or sealingagents, better adapted for use in moist environments or for underwateruse and more environmentally friendly than the present products.

PRIOR ART

[0013] It has been a well-known fact that the male three-spinedstickleback (Gasterosteus aculeatus) uses some kind of adhesivesubstance to build its typical “nest” of plant material. In FishPhysiology and Biochemistry 20: 79-85, 1999, Jakobsson et al. disclosethe stickleback nest building glue as being characterised and found toconsist almost entirely of one major protein with an apparent molecularsize of 203 kDa as determined from urinary bladder content. The proteinwas named “spiggin” after the Swedish name for stickleback, spigg.Spiggin production was shown to be inducible by 11-KT treatment and saidto be the hitherto only known protein to be induced by 11-KT.

SUMMARY OF THE INVENTION

[0014] The present inventors now make available a characterised andpurified biopolymer with many uses in analysis, medicine, and othertechnical applications as disclosed in the following description,examples and claims.

[0015] Compared to what was known prior to the priority date of thepresent application, the inventors have shown that what was thought tobe one single protein, “spiggin”, in fact is a multimerised proteincomplex comprising at least three isoforms. It is in a way unfortunate,that the previously disclosed aggregate and the surprisingly identifiedand characterised variants still carry the same name, as this may be thesource of confusion. The present inventors has however chosen to adhereto the name “spiggin” although it in the following description andclaims is used to encompass the protein complex, as well as singleisoforms, as determined by the context.

[0016] The present inventors have isolated and sequenced the nucleotidesequence, resulting in a full-length sequence, comprising approximately4.2 kb (SEQ. ID. NO. 1) and two shorter sequences, one approximately 2.2kb sequence (SEQ. ID. NO. 4) and one approximately 1.4 kb sequence (SEQ.ID. NO. 9). Further, the corresponding coding sequences have beenidentified and sequenced (SEQ. ID. NO. 2, SEQ. ID. NO. 5, and SEQ. ID.NO. 8). Finally, the inventors have isolated and purified thecorresponding proteins (SEQ. ID. NO. 3; SEQ. ID. NO. 6, and SEQ. ID. NO.7). The sequences are attached in the form of a sequence listing,prepared using PatentIn 2. 1. The sequences will be referred to by theirsequence identity numbers (SEQ. ID. NO.) and are incorporated in thedescription and claims in their entirety.

SHORT DESCRIPTION OF THE DRAWINGS

[0017] The invention will be described in closer detail below, withreference to the examples and figures, in which

[0018]FIG. 1 shows a Northern blot with the molecular mass of the cDNAsplicing variants of spiggin (Lane 1: RNA Ladder, Gibco BRL; Lane 2:Control kidney total RNA; Lane 3: 11-KT treated kidney total RNA);

[0019]FIG. 2 shows the result of a slot blot analysis of tissue specificspiggin expression within selected tissues of male three-spinedstickleback (Lane 1: fish 1; Lane 2: fish 2; Lane 3: fish 3);

[0020]FIG. 3 shows the 11-KT inducibility of the gene product in theform of a dose response analysis (% dpM/=m²) using the followingsubstances: 11-KT 4, 11-KT 20, 11-KT 100, 11-KT 500, 11-KT 2500,cortisol 100, cortisol 500, cortisol 2500, oestrogen 2500, testosterone2500, progesterone 2500, 5-α DHT 2500, and control (μg/100 μl coconutoil);

[0021]FIG. 4A shows the Coomassie brilliant blue staining of kidney andurinary bladder content on a polyacrylamid gel (SDS-PAGE, Lane 1: 11-KTtreated kidney extract; Lane 2: 11-KT treated bladder extract);

[0022]FIG. 4B shows the cross-reactivity of proteins from kidney andurinary bladder with a specific spiggin polyclonal antibody (Westernanalysis employing spiggin KTK 16 antibody, Lane 1: 11-KT treated kidneyextract; Lane 2: 11-KT treated bladder extract);

[0023]FIG. 5 shows the methyltestosterone (MT) induction of spiggin mRNAand inhibition with 1000×flutamide; and

[0024]FIG. 6 shows the spiggin mRNA levels in a Swedish pulp milllocation, proving the applicability of the inventive concept inenvironmental analysis.

DESCRIPTION OF THE INVENTION

[0025] The present invention makes available a substantially pureadhesive protein, comprising the amino acid sequence of SEQ. ID NO. 3 orfragments thereof, such as the shorter sequences of SEQ. ID. NO. 6 andSEQ. ID. NO. 7, including functionally equivalent fragments or variantsthereof. The term “functionally equivalent” is meant to encompassproteins, or polynucleotide sequences exhibiting equivalent propertieswith respect to any desired quality in question, such as the adhesiveproperty, medical properties, the capability to function as an indicatoretc.

[0026] The sequence data surprisingly reveals that the protein has aunique structure and organisation with autocatalytic sites and bindingsites for phosphorylation, carbohydrates, and lipids. Identification ofthe protein in the kidney, where it is synthesised, demonstrates that itconsists of a 130 kDa protein with smaller splicing variants (asconfirmed by nucleotide sequencing).

[0027] This also demonstrates that the previous identification of a 203kDa protein in fact was a multimer complex of at least three hereidentified and characterised proteins. Interestingly, these proteinsshow only low homology to other known proteins, with the highestsimilarity being 28% to mucins. Post-translational modifications, byglycosylation, account for a minor increase of the molecular mass of theprotein.

[0028] The invention also makes available the DNA and cDNA sequences,enclosed as SEQ. ID. NO. 1, 4, and 9; and 2, 5, and 8, respectively. Theinvention also encompasses homologous sequences, and in particularsequences exhibiting a homology greater than 50%, preferably greaterthan 70% and most preferably greater than 90%.

[0029] By operatively inserting the coding sequence in a suitablevector, and transfecting a host cell with said vector, the host cell canbe made to produce larger quantities of the protein in vitro. E. coli isone suitable host cell. It will however fall within the abilities of askilled person to select and apply a suitable vector and host cellculture. Thus, having access to the sequence data disclosed herein, thelarge scale production of the protein becomes a question of routinework.

[0030] Analytical Applications:

[0031] According to the present invention, the protein and/orinformation derived thereof, such as the sequence data, is used for thedetermination of hormone disrupting or androgenic compounds in theenvironment.

[0032] Methods:

[0033] One embodiment of the invention is a method for the detection ofandrogenic compounds in biological systems comprising the use ofspecific antibodies, for use in different protein detection systems. Inan ongoing study, the usefulness of spiggin antibodies to detectandrogenic effects in pulp mill effluent waters is investigated.Preliminary results confirm that spiggin is a good androgenic biomarker.

[0034] Another embodiment is a method for the detection of androgeniccompounds in biological systems comprising the use of the spiggin cDNAin mRNA detection systems. In an ongoing study, the usefulness ofspiggin mRNA to detect androgenic effects in pulp mill effluent watersis investigated. Preliminary results confirm that spiggin is indeed agood androgenic biomarker.

[0035] Furthermore, specific peptide sequences are to be used asstandards in antibody detection systems to allow proper quantificationof spiggin. According to one embodiment of the invention, the peptidesused for production of the antibodies are used as control standards inthe detection of androgenic compounds.

[0036] Western Blot:

[0037] One embodiment of the invention is a Western blot method todetect spiggin protein comprising the following steps:

[0038] 1) collection of a kidney or urinary bladder sample fromstickleback and mixing it with gel loading buffer,

[0039] 2) gel electrophoresis of the protein on SDS-polyacylamid gels,

[0040] 3) transfer of the protein to nylon or nitro-cellulose membrane,

[0041] 4) blocking of membrane using milk powder or equivalentsolutions,

[0042] 5) incubation with primary antibody directed against nativespiggin, synthetic spiggin peptide, or recombinant protein,

[0043] 6) washing of membrane,

[0044] 7) incubation of membrane with secondary antibody,

[0045] 8) washing of membrane, and

[0046] 9) detection of specific signal using a selection of optionaldetection systems.

[0047] ELISA:

[0048] Another embodiment of the invention is an enzyme linkedimmunosorbent assay (ELISA) comprising the following steps:

[0049] 1) collection of kidney or urinary bladder sample fromstickleback,

[0050] 2) dilution of samples in coating buffer,

[0051] 3) dilution of positive control consisting of native spiggin,synthetic peptide or recombinant protein in coating buffer,

[0052] 4) addition of samples to three wells each,

[0053] 5) addition of positive control to three wells each to create astandard curve,

[0054] 6) washing of the wells 3 times with washing buffer,

[0055] 7) addition of blocking/dilution buffer to each well,

[0056] 8) incubation at room temperature for required time,

[0057] 9) washing of wells 3 times with washing buffer,

[0058] 10) dilution of primary antibodies (against native spiggin,synthetic peptide or recombinant protein) in blocking/dilution bufferand addition to each well,

[0059] 11) incubation at an appropriate temperature for required time,

[0060] 12) washing of the wells 3 times with washing buffer,

[0061] 13) dilution of secondary antibody in blocking/dilution bufferand addition to each well,

[0062] 14) incubation at appropriate temperature for the required time,

[0063] 15) washing of the wells 3 times with washing buffer,

[0064] 16) addition of substrate solution to each well,

[0065] 17) incubation at room temperature for the required time,

[0066] 18) stopping of reaction, and

[0067] 19) measurement of signal using an optional detection method.

[0068] Kits for the detection of androgenic compounds:

[0069] Another embodiment of the invention is a kit for the detection ofandrogenic compounds, comprising at least antibodies raised against oneor several synthetic peptides designed against antigenic epitopes of theprotein, synthetic protein or recombinant protein and the specificpeptide or peptides as standard.

[0070] According to a further embodiment, the necessary components forELISA (enzyme linked immunosorbent assay) detection are also included insuch a kit. An ELISA Kit according to the invention would preferablycomprise the following:

[0071] 1) polyclonal or monoclonal antibodies raised against nativespiggin,

[0072] 2) synthetic peptides or recombinant protein designed againstantigenic epitopes of the protein,

[0073] 3) the specific synthetic peptide or recombinant protein asstandard,

[0074] 4) ELISA coating plates,

[0075] 5) coating buffer,

[0076] 6) phosphate buffered saline,

[0077] 7) washing buffer

[0078] 8) blocking buffer

[0079] 9) secondary antibody directed against rabbit for polyclonalprimary antibody or mouse for monoclonal primary antibodies,

[0080] 10) detection substrate such as horseradish peroxidase substrateor other equivalent substrate,

[0081] 11) specification chart for the primary and secondary antibodiesand the peptide standard, and

[0082] 12) a manual containing instructions for use and requirements ofadditional components.

[0083] A kit according to the present invention may also comprise onlypart of the above listed components, the necessary components beingantibodies or standards, produced using the inventive protein and/orinformation derived thereof, such as the sequence data disclosed hereinor homologous sequences, including functionally equivalent sequences.

[0084] Medical Applications:

[0085] According to further embodiments of the invention, the adhesiveprotein or protein complex is used in medical applications, for exampleas a component in wound dressings and bandages, in particular in suchapplications where the biodegradable properties of the protein areneeded.

[0086] Due to spiggin being a viscous adhesive protein that functions inwater and other moist environments it could be used e.g. for antibiotictherapy and coating of dressings. By mixing the protein with selectedpharmaceutical compounds, a slow release composition is achieved. Theadhesive properties also aid in immobilising the pharmaceutical to aspecific location of the skin or in the body of a patient, undergoingtreatment.

[0087] Since the protein has an ability to attach to surfaces, and toform an attachment between surfaces, it may be used as a tissueadhesive. The capability of attaching paper and latex samples to humanskin shows that spiggin also may be used as an adhesive for plasters,adhesives, bandages, patches and dressings etc. The protein may also beuseful in orthopaedics as a glue to keep or hold joint replacementstogether. Among other uses of this protein is the possible use as abiomechanical shock absorber due to its viscous nature. Suchbiomechanical components include artificial cartilage, intervertebraldiscs and/or parts thereof.

[0088] Technical Applications:

[0089] There is at present no glue/paste or cement that works well underwater. Since the protein has an ability to attach to surfaces, and toconnect surfaces, as tested by attaching small pieces of wood to eachother in a wet environment, it may be used as an underwateradhesive/glue. In addition to being used as an underwater glue oradhesive, the protein may also be used as an underwater sealant.

[0090] One embodiment of the present invention is the application of theadhesive protein as such, derivatives thereof or information derivedthereof for the production of a glue or an adhesive for use in moistenvironments. Moist environments in this context include both aquaticenvironments, objects and surfaces in contact with water; sea water,fresh water, high humidity, steam and/or condensation. The applicationscan be found in both natural or man-made environments and even on orwithin an animal or human body.

EXAMPLES Materials and Methods

[0091] 1. Protein Sequencing.

[0092] Amino acid analysis was performed on the 203 kDa region from geneelectrophoresis by ion-exchange chromatography as described by Jakobssonet al., Fish Physiol Biochem 20: 79-85 (1999). One potential N-terminaland several potential internal sequences were obtained. These wereSpiggin 1 (KTKEIQTY), Spiggin 2 (KAVLSIHPDFSVVK), Spiggin 3 (KENYISHK),Spiggin 4, (SYYVR) Spiggin 5 (RGTFSIR), Spiggin 6 (LYIRK), Spiggin 7(IRDPVLRK) and Spiggin 8 (TAYSWV). Of these, sequence 1, 4, 5 and 6 wereconfirmed to be spiggin following cloning of the cDNA.

[0093] 2. Hormone Treatment.

[0094] Hormones at different concentrations were dissolved in 100 ml ofcoconut oil and placed within silastic capsules (Dow Corning, internaldiameter 0.6 mm, outer diameter 1.2 mm, length 5 mm). The sealedcapsules were implanted into the abdominal cavity of each fish whichwere subsequently placed in aquaria containing 200 l of brackish water(0.5% salinity) at 17° C. under a stimulatory photoperiod of L:D 16:8.Following one month exposure the fish were sacrificed and the requiredorgans removed by dissection and stored at −70° C.

[0095] 3. RNA Extraction.

[0096] Total RNA was extracted from all tissues using TRI reagent”(Sigma). The mRNA fraction was isolated using the Poly(A) Quik′ mRNApurification kit (Stratagene). All preparations were stored at −70° C.

[0097] 4. Reverse Transcriptase—Polymerase Chain Reaction (RT-PCR).

[0098] Complementary DNA was synthesised from 0.5 mg of total RNA usingthe First Strand cDNA synthesis kit (Amersham Pharmacia Biotech). PCRwas performed with 500 ng of cDNA termplate in a final volume of 50 ml.The reaction mix contained 0.2 mM dNTPs, 5 ml 10×reaction buffer, 2.5 nMof magnesium chloride, 1.25 U of Taq DNA polymerase and 25 pmol ofSpiggin 1 (5′-CARACIAARGARATICARAC-3′) and Spiggin 3(5′-TTRTGIGAIATRTARTTYTCYTT-3′) oligonucleotides. Following an initialdenaturation step at 95° C. for 3 min, amplification was performed at95° C. for 1 min, 50° C. for 1 min, 72° C. for 1 min for 40 cycles. PCRproducts of approximately 600 bp were generated which were visualised byagarose gel electrophoresis according to Sambrook et al., MolecularCloning: A laboratory manual, 2^(nd) Ed. (1989).

[0099] 5. Sequencing of RT-PCR Products.

[0100] All generated RT-PCR products were ligated into pGEM-T′ (Promega)and transformed in competent E. coli cells according to Sambrook et al.,(1989). Positive colonies were grown under antibiotic selection overnight and recombinant plasmids were isolated using the Wizard′ Plus SVminiprep system (Promega). Cycle sequencing was performed using theThermo Sequenase v2.0 sequencing kit (Amersham Pharmacia Biotech). Thereactions were resolved on an ABI Prism″ 377 DNA sequencer (PerkinElmer) and the data obtained analysed using EditView v1.0.1 (PerkinElmer).

[0101] 6. Tissue Distribution and Dose Response Analysis.

[0102] 5 mg total RNA was combined with 90 ml of 20×SSC and 60 ml 37%formaldehyde. The final volume was adjusted to 300 ml with MQ water.Each preparation was transferred onto nylon membrane (Amersham PharmaciaBiotech) using a Minifold II Slot Blot apparatus (Schleicher & Schuell).The RNA was bound onto the membrane by cross-linking in a CL-1000 UVapparatus (UVP). Membranes were radioactively probed as described below(Paragraph 10) and the relative levels of mRNA were determined using aGS-250 Molecular Phosphoimager coupled to the Molecular Analyst (Version1:41) package (Bio Rad). All dose-response determinations were performedin triplicate and the results were expressed as the mean (±SD) for eachhormone and dose assayed.

[0103] 7. Northern Analysis.

[0104] Northern analysis of total RNA was performed according toSambrook et al., (1989) supra. Membranes were radioactively probed andvisualised as described below (Paragraph 10).

[0105] 8. Slot Blot Analysis

[0106] Total RNA was extracted from mature male tissues, from threeindividual fish, using TRI reagent® (Sigma). Aliquots of 5 mg total RNAwere mixed with denaturing solution (6×SSC, 7% (v/v) formaldehyde) andtransferred onto nylon membrane (Amersham Pharmacia Biotech) using aMinifold II Slot Blot Apparatus (Schleicher and Schuell). Membranes wereprobed using a randomly primed [a32P]-dCTP radiolabelled spiggin cDNAfragment (636 bp) that was isolated by RT-PCR and sequenced as above.Hybridisations were performed at 65° C. O/N (6×SSC, 0.1% (w/v) SDS, 100mg ml-1 tRNA and 5×Derhardt's solution). The membranes were washed for2×30 min periods at 42° C. and 65° C. in 0.1×SSC/0.1% (w/v) SDS andexposed to Hyperfilm®-MP film (Amersham Pharmacia Biotech) at −70° C.The films were visualised using a Curix 60 Filmn Developer (AGFA).

[0107] 9. cDNA Library Construction and Screening.

[0108] A cDNA library was generated from the poly-A fraction ofstickleback kidney RNA which was isolated as described previously(Paragraph 3). The library was constructed in Lambda ZAP Express′(Stratagene). Screening was performed according to Sambrook et al.,(1989) supra, using radioactive probing as described below (Paragraph10). Several individual clones were identified following four rounds.These were isolated according to the Lambda ZAP Express′ instructionmanual and sequenced as described below (Paragraph 9).

[0109] 10. Sequencing of Library Isolated Clones.

[0110] Initial sequencing was done as described in Paragraph 5 toconfirm the identity of individual spiggin cDNA clones. The selectedclones were thereafter sequenced by Cybergene AB, Huddinge, Sweden (100%Patent Sequencing).

[0111] 11. Radioactive Probing.

[0112] All membranes were probed using a 600 bp [a32P]-dCTPradiolabelled cDNA fragment of spiggin which was previously isolated byRT-PCR (Paragraph 4). Hybridisation was performed at 65° C. O/N in6×SSC, 0.1% (w/v) SDS, 100 mg ml-1 tRNA and 5×Denhardt's solution (0.1%(w/v) BSA, 0.1% (w/v) ficoll, 0.1% (w/v) polyvinylpyrrolidone). Themembranes were washed for 2×30 min periods at 42° C. and 65° C. in1×SSC, 0.1% (w/v) SDS and exposed to Hyperfilm″-MP film (AmershamPharmacia Biotech) at −70° C. O/N. The films were visualised using aCurix 60 film developer (AGFA).

[0113] 12. Production of Polyclonal Antibodies.

[0114] Polyclonal antibodies were generated by Agri Sera AB. Thefollowing peptides were employed: Spiggin KTK 16 (KTIEIQTYTSRTFGS-C) andSpiggin HRD 16 (HIRDELIRDSHLHDHR-C) which correspond to the N-terminalsequence and amino acid sequences at positions 149-164, respectively ofthe spiggin protein.

[0115] 13. SDS-PAGE.

[0116] Protein samples were resolved by SDS-PAGE at a constant currentof 50 mA for 1 h according to Laemnmli (1970) using 10% and 5%Tris-glycine running and stacking gels, respectively. Proteins weresubsequently visualised by staining with Coomasie brilliant blue, orsilver nitrate according to Sambrook et al., (1989) supra.

[0117] 14. Western Blot Analysis.

[0118] Proteins were transferred onto nitro-cellulose membrane (AmershamPharmacia Biotech) following SDS-PAGE using the Trans-Blot′ Semi DryElectrophoretic Transfer Cell (Bio Rad) at 15 V for 30 min in a bufferwhich consisted of 48 mM Tris, 39 mM glycine and 20% methanol (pH 9.2).Immunodetection was performed according to Sambrook et al., (1989) usingsera raised against either Spiggin KTK 16 or Spiggin HRD 16 as primaryantibodies (Paragraph 11) and rabbit immunoglobulin HRP conjugate (DAKO)as secondary antibody, respectively. Proteins were visualised using theHRP substrate kit (Bio Rad).

[0119] 15. Adhesive Properties.

[0120] Spiggin protein obtained from urinary bladders was tested for itsability to hold together biological material such as pieces of wood.This ability was tested by holding together the pieces and thendetermining if they would remain attached. The ability to adhere tohuman skin was tested according to the following protocol:

[0121] The male stickleback has a high content of spiggin in its urinarybladder during the breeding season. Three male stickleback werecollected and their urinary bladder content extracted. This urinarybladder content will below be called “spiggin”. Spiggin was compared toa solution of bovine serum albumin (BSA), mineral oil, and water for itsability to attach 1 cm² samples of KIM® wipe paper or latex (cut from asurgical glove) to human skin. The site of attachment was the lower sideof the forearm of the testperson. The experiment was performed thrice.An initial test was performed using only spiggin and water and thesamples observed for 60 minutes (Experiment I). This was followed by twoexperiments where the samples were observed for 20 minutes (ExperimentsII and III). At the end of each experiment, the samples were removedfrom the skin using a fine forceps. Attachment was determined as none(−), very weak (+/−), weak (+), strong (++), and very strong (+++). If asample detached on its own, the time to detachment was registered.

[0122] 16. Methyltestosterone Induction of Spiggin mRNA

[0123] Juvenile three spined sticklebacks, with a body weight ofapproximately 100 mg, were exposed to different doses (100 pM, 10 nM and1 uM) of methyltestosterone or to a combination of 10 nMmethyltestosterone and 10 uM flutamide (an antiandrogen). After 10 daysthe fish were sacrificed. Whole fish were homogenized and spiggin mRNAlevels were determined using slot blot analysis. The results arepresented in FIG. 5.

[0124] In a separate experiment juvenile three spined stickleback wereexposed to water samples collected from a Swedish river and a pulp millin this river. The fish were exposed to 10×diluted pulpmill and riveroutlet water and undiluted incoming river water for 10 days. As acontrol a separate group was exposed to undiluted spring water. Wholefish were analysed for spiggin mRNA content using slot blot analysis.The methyltestosterone exposure was plotted to create a standard curve.The spiggin levels in the water exposed fish were compared to thestandard curve and presented as nM methyltestosterone equivalents. Theresults are presented in FIG. 6.

[0125] Results

[0126] Sequencing of the stickleback kidney cDNA library revealed thepresence of at least three splicing variants of the spiggin protein.These variants are encoded for by a 4.2 kb (SEQ ID NO. 1), a 2.2 kb (SEQID NO. 4), and a 1.6 kb (SEQ ID NO. 8) mRNA. Northern blot analysis of11KT induced stickleback kidney (FIG. 2) indicates that there are atleast three different splicing variants, with the largest being the 4.2kb mRNA that has been cloned and the 1.6 kb mRNA being the smallest.

[0127] The stickleback spiggin glue protein is exclusively expressed inthe kidney of sexually maturing male stickleback (FIG. 2) and is onlyinducible by 11-ketotestosterone (FIG. 3).

[0128] The largest cloned cDNA codes for a 910 amino acid protein with amolecular mass of 103 IDa, which surprisingly was found to beapproximately 50% of the previously determined size of spiggin inurinary bladder. The spiggin protein has been estimated to 203 kDa inurine and has been shown to have some glycosylation resulting in a fewpercent reduction in molecular mass.

[0129] To determine the reason for the discrepancy in molecular mass thepresent inventors produced 2 polyclonal antibodies. One was directedagainst the N-terminal region KTKIEQTYTSRTFGS (amino acid 25-39)following the leader sequence, and the other was directed against anantigenic epitope located between amino acid 173 and 188,HRDELIRDSHLHDHRC. Using both these antibodies to detect spiggin inkidney and urinary bladder demonstrated that the antibodies detected the203 kDa urinary product while this product was not present in kidney(FIG. 4b).

[0130] The largest protein detected in kidney was approximately 130 kDa,which is in good agreement with the deduced protein encoded by the 4.2kb mRNA. Several smaller components, probably reflecting both thesplicing variants (51 kDa and 90 kDa) and degradation produces were alsodetected on the Western blots. Detection of spiggin using Coomasiebrilliant blue staining also indicate that the 203 kDa protein is notpresent in kidney but is a modified product, probably a dimer, thatappears first in the urinary bladder (FIG. 4a).

[0131] Together these results show that the spiggin gene encodes for atleast 3 splicing variants and that the largest of these has anunmodified molecular mass of 103 kDa. Furthermore This protein appearsas a 203 kDa product in urinary bladder, probably as a result ofmultimerisation.

[0132] The spiggin protein has also been tested for its ability to holdpieces of wood together in a wet environment. The present inventorsfound that addition of spiggin resulted in attachment of the pieces.This is in line with the function of spiggin in nature where it is usedby the male stickleback to glue together biological material to form anest.

[0133] The experiments evaluating the ability of spiggin to attachshowed promising results. Spiggin was able to firmly attach a piece ofKim wipe paper in all three tests. Spiggin attached latex had a fluidattachment for about 10 min before drying out and attaching the latex.The results are presented in the table below: TABLE 1 Ability of spigginto attach sample to human skin Solution KIM Wipe LATEX Experiment IWater + 10 minutes Spiggin + + + + Experiment II Water + +/− Mineral oil− 15 minutes Bovine serum albumin − +/− Spiggin + + + + Experiment IIIWater 4 minutes +/− Mineral oil 3 minutes 10 minutes Bovine serumalbumin +/− +/− Spiggin + + + +

[0134] The ability of spiggin to function as an androgenic marker wastested, both using different doses (100 pM, 10 nM and 1 uM) ofmethyltestosterone, a combination of 10 nM methyltestosterone and 10 uMflutamide (an antiandrogen), and diluted pulpmill outlet water.

[0135] In experiments evaluating the ability of spiggin to beupregulated by androgenic compounds via water it was found thatmethyltestosterone down to 100 pM was effective at inducing spigginmRNA. Furthermore, the androgen specificity of the system was confirmedby the inhibiting ability of flutamide (an antiandrogen) when addedtogether with to methyltestosterone.

[0136] Confirmation that androgenic responses are present in theenvironment was obtained by testing water from a river and a pulpmill.The river sampling point showed weak androgenic effects comparable to a200 pM methyltestosterone exposure. This water is down stream of a majorsewage treatment plant. Higher levels of spiggin mRNA induction wasobserved within the pulp mill with levels reaching 1400 pMmethyltestosterone equivalents. Finally, the ability of the pulp mill topurify the outlet water was observed by the lower androgenicity of theRiver outlet water. Together these studies demonstrate that spiggin is ahighly sensitive biomarker for androgenic effects. The results arepresented in FIGS. 5 and 6.

[0137] Although the invention has been described with regard to itspreferred embodiments, which constitute the best mode presently known tothe inventors, it should be understood that various changes andmodifications as would be obvious to one having the ordinary skill inthis art may be made without departing from the scope of the inventionwhich is set forth in the claims appended hereto.

1 9 1 4191 DNA Gasterosteus aculeatus 1 catcaggcag catgacaacc cagcgatggattctggcatt ctgcctttca ctggcctcag 60 tttttggcac agtggaattg ttcaagaccaaagaaattca gacttacact tgcaggacat 120 ttggcagcgg gatcgtccag ccttttaagggggagagtta ctatgttcgg tccgactgcc 180 cgttcaaact cacaagcttc aacgtcaaccggggggaata ttctgtcacc atacggcgag 240 gtcacaacgg gctgttggtc caagtcgagatcatcatcaa caaagtcaca acacttttgc 300 agaatggcca catcctagtg cagaacaaaagtgtttcact tccatacgac cacacctacc 360 agcatatctt taaatacggc atctacactagactgaggag ctcgctgctt ccttttactg 420 tcacctggca caatgtacat gggggaataaactctctgtg ggtgacactg gagtcggagc 480 tgtgcaccga catgtgtgga ctgtgtggaaaacaaaacgt tgcaggccac agggacgagt 540 tgatcagaga cagcaagctt catgaccacagatgtaaaat cagagatcct gtgttgcgaa 600 aaaatcacat atgccgtcga ttctttctgaaaaccaagaa ctgtctgcaa gacaacaatt 660 ctcactatca ccgactctgt aaagagaacatttgtggctt tgaaaacagc cagagcatct 720 tctgtccttt cttccaagaa gttgcaagccagtgtaacca atcaagaatc aaccgatttt 780 ggagacgttt aaccaaatgt gcgaagccgaggtgtccagg agacctgatt tatgagaaaa 840 aaggtccagc atttattccc agctgctccaacccgaaccc tgcacccttc taccaggaac 900 tcactgaaac ctgttcctgt ccagagggtaaagttttgaa taattgtgaa aagggctatc 960 gctgtatacc taaatccagc tgctcttgtgagtttgctgg caagacctac ggaaacggag 1020 aaatacggag ttccaggtgt cagtcatgtacgtgtgatgg tgggaaatgg cgatgctcag 1080 agaacttttg ccacagaaga tgtgtcattgaaggccagtt tgtgacaaca ttcgatggaa 1140 aacaatatgt cctccctaac aaatgtttatatgtggcttc aaagggtccc aactggataa 1200 taataataga gttttcgcaa aaaaacctctacattagaaa ggttacggtt cagctcatgg 1260 aggaactgtt tgtattcaaa aacaacaaggttttgtttga tggacaggag atccctgaat 1320 tccatttttc tggtcatgct caggtttactgggtgtcctc catgttcgtc caggtccaca 1380 cgaccattgg tataaacttc caaattcagatgtcccctga aatccatctg ttcatcgacg 1440 cacctgaccc ctccagtgac aagattaaaggtctttgtgg caatagcaac agtgacacca 1500 cagatgactt caccaccaac agcgggatcattgagaactc agctaaacca tttgctatgt 1560 cctggagttt gcttaattgt ttcggtaacatacccaccac ctgcaccaac ctggagaatg 1620 agaattatgc tcatgaaaag tgtgcagtgttaaaccaacc aactgggata tttgctaagt 1680 gccaccctca tatcccaact gattactactacacggcttg catccaaaga atatgtaact 1740 ctgccggaag tcggagacag ggcttgtgtattggtctggc cagctacgcc aaagcctgcg 1800 ccggtgttgg tgttgtaatt ggtgactggaggagaatcac gggctgcgat ctgaaatgcc 1860 agaagaacca agaattctcc tacagcatgcatacatgcaa ccgcacatgc aattctctga 1920 ccggccatga catccgctgt ggtatgaatgacgatgctgt ggagggctgt ggctgtccgg 1980 agggaactca cctgaaccaa ggacagacctgttgcccaaa ggaagagtgt ggttgtattt 2040 actacggtgg tattgcagcc ccggggcctgttgttatcgc cggacaaaag tgcgactgca 2100 agaatgggat actgaactgc ttgccgaattgtgattgcag aaatgggaag gtgtgtgtca 2160 gttgctctga gggccaacat aagagggttcagaagacctg tgactatatt agcaaaccaa 2220 agggtaccag ggagaactgt aagagtggctgttactgtcc agatcaccag tatgaagatc 2280 accatgggaa ctgtgtttca ctcgatgattgcacctgtgt gttcagtggc aaagcattca 2340 aagctggaca gcaagttacc agcaactgtaaaacatgtac ctgttatcgt ggtcagtggc 2400 actgcattga gaagccctgc ccgggacagtgccaagtcta cggaaatgga cactaccaga 2460 cctttgactc caaatggttc cgcttttctggacaatgtct gtacacactt gtgcaggatt 2520 cctgtgacat gagaagaggc accttctctatcagagtgga gagtgtcccc tgctgtgagg 2580 aggtgctcac ctgctctcgc aacatcatccttgacctgaa gggccaagtc accctgacgc 2640 tgagagacat gcaggtgacc agacgcctccatgaaggctg gactgggcag gatgattcac 2700 tttactcaat acacactttg ggactttaccaacatgcgct ctgacgacag agtcaacaat 2760 tcaatgccca gcatggccac gacagcagtgggcgtgtcca tcttcctgct gaccaccacc 2820 gcctgcctgg tggtcaagtc ccgccttatcccgtgtcaat cacacagccg gcgctcttca 2880 gaccagctgg acctgatggt ggacggacttcccgtctccc tcccctccta cgaggaggcg 2940 atgtacagca gctgggggca gcgcctccccgccttctcag cgccgggggg ccccacccag 3000 ctcctactgg ctcaagaggc tcccagctgtcacccagcgg ctctcaacaa tcaagacagc 3060 agcaaccgcc cccccctgcc cagccccgacaacccgccgc ccccgtacga ggaggtgcag 3120 tcgtcacaac atagggacag gatgggggacggggacatcc ggccgctccg cgtcgcgttt 3180 tcagatgaca aggacacttg attgacagactgcagggttt acatgtgatt acgtgtttag 3240 caatgaagga ttttgattga cagctgcggtgtaaaaatac atccagtact gcactttttc 3300 tgactggctg cagctcacca gcatcacatgctaatttaga aaaagctgat aagctctgat 3360 tggccggcag tgaggtcaca aagacctgaaccatttctac gggaggtatt gattgtaaag 3420 tgtgtctatt gaatgtgaga caagggcacttgtgctttat gccgaaacct ttagctcccg 3480 aatgccttat ttttagtgtg atgacagtacgttttattgg aattatgagt gtgtttctgg 3540 agtcggtgag agtgtggatt gtgctgttgcagtacaaaaa tcctgtgcgt ttccacggca 3600 actgactcca tctctatctg aatctacctttggtaaagtg tttctcttgg tggtgttgat 3660 ttaaagccag cgtggtgcac agtgaaggtactaattacat gctcgtgtgc ggacactagg 3720 acgatagatt gtgacaaaca agcaccattttgtgatattt attttgtgta tatcaaatat 3780 ttagagaagt tacaatgatc ggatgtgttcatttactggt gagtgaagga aaattttata 3840 ttttgattaa ttgaaatgtc taatcaagagaatataacat tattgtgcat ttgctccatc 3900 agtggagact cacaaagacg acagttttcattctggacca aataacccca aagtgcgtgc 3960 gtgcgtgcgt gcgcatgcat cttcacttcaccaggctact tccccctcta gcctctgcag 4020 agcgaccaac gcctttacag accttgaatgtattttatgt ttcatattgt ggcaacaaat 4080 cctaactgga ttgtaatatt taccaaaagattatttgtac atttgtgtta ttgcacagaa 4140 ttatatgatc aataaatgtt gacgtttgaaaaaaaaaaaa aaaaaaaaaa a 4191 2 2733 DNA Gasterosteus aculeatus 2atgacaaccc agcgatggat tctggcattc tgcctttcac tggcctcagt ttttggcaca 60gtggaattgt tcaagaccaa agaaattcag acttacactt gcaggacatt tggcagcggg 120atcgtccagc cttttaaggg ggagagttac tatgttcggt ccgactgccc gttcaaactc 180acaagcttca acgtcaaccg gggggaatat tctgtcacca tacggcgagg tcacaacggg 240ctgttggtcc aagtcgagat catcatcaac aaagtcacaa cacttttgca gaatggccac 300atcctagtgc agaacaaaag tgtttcactt ccatacgacc acacctacca gcatatcttt 360aaatacggca tctacactag actgaggagc tcgctgcttc cttttactgt cacctggcac 420aatgtacatg ggggaataaa ctctctgtgg gtgacactgg agtcggagct gtgcaccgac 480atgtgtggac tgtgtggaaa acaaaacgtt gcaggccaca gggacgagtt gatcagagac 540agcaagcttc atgaccacag atgtaaaatc agagatcctg tgttgcgaaa aaatcacata 600tgccgtcgat tctttctgaa aaccaagaac tgtctgcaag acaacaattc tcactatcac 660cgactctgta aagagaacat ttgtggcttt gaaaacagcc agagcatctt ctgtcctttc 720ttccaagaag ttgcaagcca gtgtaaccaa tcaagaatca accgattttg gagacgttta 780accaaatgtg cgaagccgag gtgtccagga gacctgattt atgagaaaaa aggtccagca 840tttattccca gctgctccaa cccgaaccct gcacccttct accaggaact cactgaaacc 900tgttcctgtc cagagggtaa agttttgaat aattgtgaaa agggctatcg ctgtatacct 960aaatccagct gctcttgtga gtttgctggc aagacctacg gaaacggaga aatacggagt 1020tccaggtgtc agtcatgtac gtgtgatggt gggaaatggc gatgctcaga gaacttttgc 1080cacagaagat gtgtcattga aggccagttt gtgacaacat tcgatggaaa acaatatgtc 1140ctccctaaca aatgtttata tgtggcttca aagggtccca actggataat aataatagag 1200ttttcgcaaa aaaacctcta cattagaaag gttacggttc agctcatgga ggaactgttt 1260gtattcaaaa acaacaaggt tttgtttgat ggacaggaga tccctgaatt ccatttttct 1320ggtcatgctc aggtttactg ggtgtcctcc atgttcgtcc aggtccacac gaccattggt 1380ataaacttcc aaattcagat gtcccctgaa atccatctgt tcatcgacgc acctgacccc 1440tccagtgaca agattaaagg tctttgtggc aatagcaaca gtgacaccac agatgacttc 1500accaccaaca gcgggatcat tgagaactca gctaaaccat ttgctatgtc ctggagtttg 1560cttaattgtt tcggtaacat acccaccacc tgcaccaacc tggagaatga gaattatgct 1620catgaaaagt gtgcagtgtt aaaccaacca actgggatat ttgctaagtg ccaccctcat 1680atcccaactg attactacta cacggcttgc atccaaagaa tatgtaactc tgccggaagt 1740cggagacagg gcttgtgtat tggtctggcc agctacgcca aagcctgcgc cggtgttggt 1800gttgtaattg gtgactggag gagaatcacg ggctgcgatc tgaaatgcca gaagaaccaa 1860gaattctcct acagcatgca tacatgcaac cgcacatgca attctctgac cggccatgac 1920atccgctgtg gtatgaatga cgatgctgtg gagggctgtg gctgtccgga gggaactcac 1980ctgaaccaag gacagacctg ttgcccaaag gaagagtgtg gttgtattta ctacggtggt 2040attgcagccc cggggcctgt tgttatcgcc ggacaaaagt gcgactgcaa gaatgggata 2100ctgaactgct tgccgaattg tgattgcaga aatgggaagg tgtgtgtcag ttgctctgag 2160ggccaacata agagggttca gaagacctgt gactatatta gcaaaccaaa gggtaccagg 2220gagaactgta agagtggctg ttactgtcca gatcaccagt atgaagatca ccatgggaac 2280tgtgtttcac tcgatgattg cacctgtgtg ttcagtggca aagcattcaa agctggacag 2340caagttacca gcaactgtaa aacatgtacc tgttatcgtg gtcagtggca ctgcattgag 2400aagccctgcc cgggacagtg ccaagtctac ggaaatggac actaccagac ctttgactcc 2460aaatggttcc gcttttctgg acaatgtctg tacacacttg tgcaggattc ctgtgacatg 2520agaagaggca ccttctctat cagagtggag agtgtcccct gctgtgagga ggtgctcacc 2580tgctctcgca acatcatcct tgacctgaag ggccaagtca ccctgacgct gagagacatg 2640caggtgacca gacgcctcca tgaaggctgg actgggcagg atgattcact ttactcaata 2700cacactttgg gactttacca acatgcgctc tga 2733 3 910 PRT Gasterosteusaculeatus 3 Met Thr Thr Gln Arg Trp Ile Leu Ala Phe Cys Leu Ser Leu AlaSer 1 5 10 15 Val Phe Gly Thr Val Glu Leu Phe Lys Thr Lys Glu Ile GlnThr Tyr 20 25 30 Thr Cys Arg Thr Phe Gly Ser Gly Ile Val Gln Pro Phe LysGly Glu 35 40 45 Ser Tyr Tyr Val Arg Ser Asp Cys Pro Phe Lys Leu Thr SerPhe Asn 50 55 60 Val Asn Arg Gly Glu Tyr Ser Val Thr Ile Arg Arg Gly HisAsn Gly 65 70 75 80 Leu Leu Val Gln Val Glu Ile Ile Ile Asn Lys Val ThrThr Leu Leu 85 90 95 Gln Asn Gly His Ile Leu Val Gln Asn Lys Ser Val SerLeu Pro Tyr 100 105 110 Asp His Thr Tyr Gln His Ile Phe Lys Tyr Gly IleTyr Thr Arg Leu 115 120 125 Arg Ser Ser Leu Leu Pro Phe Thr Val Thr TrpHis Asn Val His Gly 130 135 140 Gly Ile Asn Ser Leu Trp Val Thr Leu GluSer Glu Leu Cys Thr Asp 145 150 155 160 Met Cys Gly Leu Cys Gly Lys GlnAsn Val Ala Gly His Arg Asp Glu 165 170 175 Leu Ile Arg Asp Ser Lys LeuHis Asp His Arg Cys Lys Ile Arg Asp 180 185 190 Pro Val Leu Arg Lys AsnHis Ile Cys Arg Arg Phe Phe Leu Lys Thr 195 200 205 Lys Asn Cys Leu GlnAsp Asn Asn Ser His Tyr His Arg Leu Cys Lys 210 215 220 Glu Asn Ile CysGly Phe Glu Asn Ser Gln Ser Ile Phe Cys Pro Phe 225 230 235 240 Phe GlnGlu Val Ala Ser Gln Cys Asn Gln Ser Arg Ile Asn Arg Phe 245 250 255 TrpArg Arg Leu Thr Lys Cys Ala Lys Pro Arg Cys Pro Gly Asp Leu 260 265 270Ile Tyr Glu Lys Lys Gly Pro Ala Phe Ile Pro Ser Cys Ser Asn Pro 275 280285 Asn Pro Ala Pro Phe Tyr Gln Glu Leu Thr Glu Thr Cys Ser Cys Pro 290295 300 Glu Gly Lys Val Leu Asn Asn Cys Glu Lys Gly Tyr Arg Cys Ile Pro305 310 315 320 Lys Ser Ser Cys Ser Cys Glu Phe Ala Gly Lys Thr Tyr GlyAsn Gly 325 330 335 Glu Ile Arg Ser Ser Arg Cys Gln Ser Cys Thr Cys AspGly Gly Lys 340 345 350 Trp Arg Cys Ser Glu Asn Phe Cys His Arg Arg CysVal Ile Glu Gly 355 360 365 Gln Phe Val Thr Thr Phe Asp Gly Lys Gln TyrVal Leu Pro Asn Lys 370 375 380 Cys Leu Tyr Val Ala Ser Lys Gly Pro AsnTrp Ile Ile Ile Ile Glu 385 390 395 400 Phe Ser Gln Lys Asn Leu Tyr IleArg Lys Val Thr Val Gln Leu Met 405 410 415 Glu Glu Leu Phe Val Phe LysAsn Asn Lys Val Leu Phe Asp Gly Gln 420 425 430 Glu Ile Pro Glu Phe HisPhe Ser Gly His Ala Gln Val Tyr Trp Val 435 440 445 Ser Ser Met Phe ValGln Val His Thr Thr Ile Gly Ile Asn Phe Gln 450 455 460 Ile Gln Met SerPro Glu Ile His Leu Phe Ile Asp Ala Pro Asp Pro 465 470 475 480 Ser SerAsp Lys Ile Lys Gly Leu Cys Gly Asn Ser Asn Ser Asp Thr 485 490 495 ThrAsp Asp Phe Thr Thr Asn Ser Gly Ile Ile Glu Asn Ser Ala Lys 500 505 510Pro Phe Ala Met Ser Trp Ser Leu Leu Asn Cys Phe Gly Asn Ile Pro 515 520525 Thr Thr Cys Thr Asn Leu Glu Asn Glu Asn Tyr Ala His Glu Lys Cys 530535 540 Ala Val Leu Asn Gln Pro Thr Gly Ile Phe Ala Lys Cys His Pro His545 550 555 560 Ile Pro Thr Asp Tyr Tyr Tyr Thr Ala Cys Ile Gln Arg IleCys Asn 565 570 575 Ser Ala Gly Ser Arg Arg Gln Gly Leu Cys Ile Gly LeuAla Ser Tyr 580 585 590 Ala Lys Ala Cys Ala Gly Val Gly Val Val Ile GlyAsp Trp Arg Arg 595 600 605 Ile Thr Gly Cys Asp Leu Lys Cys Gln Lys AsnGln Glu Phe Ser Tyr 610 615 620 Ser Met His Thr Cys Asn Arg Thr Cys AsnSer Leu Thr Gly His Asp 625 630 635 640 Ile Arg Cys Gly Met Asn Asp AspAla Val Glu Gly Cys Gly Cys Pro 645 650 655 Glu Gly Thr His Leu Asn GlnGly Gln Thr Cys Cys Pro Lys Glu Glu 660 665 670 Cys Gly Cys Ile Tyr TyrGly Gly Ile Ala Ala Pro Gly Pro Val Val 675 680 685 Ile Ala Gly Gln LysCys Asp Cys Lys Asn Gly Ile Leu Asn Cys Leu 690 695 700 Pro Asn Cys AspCys Arg Asn Gly Lys Val Cys Val Ser Cys Ser Glu 705 710 715 720 Gly GlnHis Lys Arg Val Gln Lys Thr Cys Asp Tyr Ile Ser Lys Pro 725 730 735 LysGly Thr Arg Glu Asn Cys Lys Ser Gly Cys Tyr Cys Pro Asp His 740 745 750Gln Tyr Glu Asp His His Gly Asn Cys Val Ser Leu Asp Asp Cys Thr 755 760765 Cys Val Phe Ser Gly Lys Ala Phe Lys Ala Gly Gln Gln Val Thr Ser 770775 780 Asn Cys Lys Thr Cys Thr Cys Tyr Arg Gly Gln Trp His Cys Ile Glu785 790 795 800 Lys Pro Cys Pro Gly Gln Cys Gln Val Tyr Gly Asn Gly HisTyr Gln 805 810 815 Thr Phe Asp Ser Lys Trp Phe Arg Phe Ser Gly Gln CysLeu Tyr Thr 820 825 830 Leu Val Gln Asp Ser Cys Asp Met Arg Arg Gly ThrPhe Ser Ile Arg 835 840 845 Val Glu Ser Val Pro Cys Cys Glu Glu Val LeuThr Cys Ser Arg Asn 850 855 860 Ile Ile Leu Asp Leu Lys Gly Gln Val ThrLeu Thr Leu Arg Asp Met 865 870 875 880 Gln Val Thr Arg Arg Leu His GluGly Trp Thr Gly Gln Asp Asp Ser 885 890 895 Leu Tyr Ser Ile His Thr LeuGly Leu Tyr Gln His Ala Leu 900 905 910 4 2197 DNA Gasterosteusaculeatus 4 gcagcatgac aacccagcga tggattctgg cattctgcct ttcactggcctcagtttttg 60 gcacagtgga attgttcaag accaaagaaa ttcagactta cacttgcaggacatttggca 120 gcgggatcgt ccagcctttt aagggggaga gttactatgt tcggtccgactgcccgttca 180 aactcacaag cttcaacgtc aaccgggggg aatattctgt caccatacggcgaggtcaca 240 acgggctgtt ggtccaagtc gagatcatca tcaacaaagt cacaacacttttgcagaatg 300 gccacatcct agtgcagaac aaaagtgttt cacttccata cgaccacacctaccagcata 360 tctttaaata cggcatctac actagactga ggagctcgct gcttccttttactgtcacct 420 ggcacaatgt acatggggga ataaactctc tgtgggtgac actggagtcggagctgtgca 480 ccgacatgtg tggactgtgt ggaaaacaaa acgttgcagg ccacagggacgagttgatca 540 gagacagcaa gcttcatgac cacagatgta aaatcagaga tcctgtgttgcgaaaaaatc 600 acatatgccg tcgattcttt ctgaaaacca agaactgtct gcaagacaacaattctcact 660 atcaccgact ctgtaaagag aacatttgtg gctttgaaaa cagccagagcatcttctgtc 720 ctttcttcca agaagttgca agccagtgta accaatcaag aatcaaccgattttggagac 780 gtttaaccaa atgtgcgaag ccgaggtgtc caggagacct gatttatgagaaaaaaggtc 840 cagcatttat tcccagctgc tccaacccga accctgcacc cttctaccaggaactcactg 900 aaacctgttc ctgtccagag ggtaaagttt tgaataattg tgaaaagggctatcgctgta 960 tacctaaatc cagctgctct tgtgagtttg ctggcaagac ctacggaaacggagaaatac 1020 ggagttccag gtgtcagtca tgtacgtgtg atggtgggaa atggcgatgctcagagaact 1080 tttgccacag aagatgtgtc attgaaggcc agtttgtgac aacattcgatggaaaacaat 1140 atgtcctccc taacaaatgt ttatatgtgg cttcaaaggg tcccaactggataataataa 1200 tagagttttc gcaaaaaaac ctctacatta gaaaggttac ggttcagctcatggaggaac 1260 tgtttgtatt caaaaacaac aaggttttgt ttgatggaca ggagatccctgaattccatt 1320 tttctggtca tgctcaggtt tactgggtgt cctccatgtt cgtccaggtccacacgacca 1380 ttggtataaa cttccaaatt cagatgtccc ctgaaatcca tctgttcatcgacgcacctg 1440 acccctccag tgacaagatt aaaggtcttt gtggcaatag caacagtgacaccacagatg 1500 acttcaccac caacagcggg atcattgaga actcagctaa accatttgctatgtcctgga 1560 gtttgcttaa ttgtttcggt aacataccca ccacctgcac caacctggagaatgagaatt 1620 atgctcatga aaagtgtgca gtgttaaacc aaccaactgg gatatttgctaagtgccacc 1680 ctcatatccc aactgattac tactacacgg cttgcatcca aagaatatgtaactctgccg 1740 gaagtcggag acagggcttg tgtattggtc tggccagcta cgccaaagcctgcgccggtg 1800 ttggtgttgt aattggtgac tggcttgagc atcaaggtag gccataatctgtgtgtgcat 1860 gcacagaaca attgctgtca ggagaagacc tcggagacaa aaacgctctccgcgatatgc 1920 taccacccta taaccaacag aagagaggaa aaaacgttca cctacaagcacatcacctct 1980 tgtgagtgca gactgtgtaa cacacaacac tgaaaaaatg ttgttagcgcagccattttc 2040 atcctgttcc cttcagattt ctaaatcaaa tcaaaatata taggactcaaattgttttta 2100 gtgattcatt ttatttcttc tgcaaagaaa ttttctgatc tgcttttagtcaaataaaag 2160 taatcattgt ctactcacca aaaaaaaaaa aaaaaaa 2197 5 1842 DNAGasterosteus aculeatus 5 atgacaaccc agcgatggat tctggcattc tgcctttcactggcctcagt ttttggcaca 60 gtggaattgt tcaagaccaa agaaattcag acttacacttgcaggacatt tggcagcggg 120 atcgtccagc cttttaaggg ggagagttac tatgttcggtccgactgccc gttcaaactc 180 acaagcttca acgtcaaccg gggggaatat tctgtcaccatacggcgagg tcacaacggg 240 ctgttggtcc aagtcgagat catcatcaac aaagtcacaacacttttgca gaatggccac 300 atcctagtgc agaacaaaag tgtttcactt ccatacgaccacacctacca gcatatcttt 360 aaatacggca tctacactag actgaggagc tcgctgcttccttttactgt cacctggcac 420 aatgtacatg ggggaataaa ctctctgtgg gtgacactggagtcggagct gtgcaccgac 480 atgtgtggac tgtgtggaaa acaaaacgtt gcaggccacagggacgagtt gatcagagac 540 agcaagcttc atgaccacag atgtaaaatc agagatcctgtgttgcgaaa aaatcacata 600 tgccgtcgat tctttctgaa aaccaagaac tgtctgcaagacaacaattc tcactatcac 660 cgactctgta aagagaacat ttgtggcttt gaaaacagccagagcatctt ctgtcctttc 720 ttccaagaag ttgcaagcca gtgtaaccaa tcaagaatcaaccgattttg gagacgttta 780 accaaatgtg cgaagccgag gtgtccagga gacctgatttatgagaaaaa aggtccagca 840 tttattccca gctgctccaa cccgaaccct gcacccttctaccaggaact cactgaaacc 900 tgttcctgtc cagagggtaa agttttgaat aattgtgaaaagggctatcg ctgtatacct 960 aaatccagct gctcttgtga gtttgctggc aagacctacggaaacggaga aatacggagt 1020 tccaggtgtc agtcatgtac gtgtgatggt gggaaatggcgatgctcaga gaacttttgc 1080 cacagaagat gtgtcattga aggccagttt gtgacaacattcgatggaaa acaatatgtc 1140 ctccctaaca aatgtttata tgtggcttca aagggtcccaactggataat aataatagag 1200 ttttcgcaaa aaaacctcta cattagaaag gttacggttcagctcatgga ggaactgttt 1260 gtattcaaaa acaacaaggt tttgtttgat ggacaggagatccctgaatt ccatttttct 1320 ggtcatgctc aggtttactg ggtgtcctcc atgttcgtccaggtccacac gaccattggt 1380 ataaacttcc aaattcagat gtcccctgaa atccatctgttcatcgacgc acctgacccc 1440 tccagtgaca agattaaagg tctttgtggc aatagcaacagtgacaccac agatgacttc 1500 accaccaaca gcgggatcat tgagaactca gctaaaccatttgctatgtc ctggagtttg 1560 cttaattgtt tcggtaacat acccaccacc tgcaccaacctggagaatga gaattatgct 1620 catgaaaagt gtgcagtgtt aaaccaacca actgggatatttgctaagtg ccaccctcat 1680 atcccaactg attactacta cacggcttgc atccaaagaatatgtaactc tgccggaagt 1740 cggagacagg gcttgtgtat tggtctggcc agctacgccaaagcctgcgc cggtgttggt 1800 gttgtaattg gtgactggct tgagcatcaa ggtaggccataa 1842 6 613 PRT Gasterosteus aculeatus 6 Met Thr Thr Gln Arg Trp IleLeu Ala Phe Cys Leu Ser Leu Ala Ser 1 5 10 15 Val Phe Gly Thr Val GluLeu Phe Lys Thr Lys Glu Ile Gln Thr Tyr 20 25 30 Thr Cys Arg Thr Phe GlySer Gly Ile Val Gln Pro Phe Lys Gly Glu 35 40 45 Ser Tyr Tyr Val Arg SerAsp Cys Pro Phe Lys Leu Thr Ser Phe Asn 50 55 60 Val Asn Arg Gly Glu TyrSer Val Thr Ile Arg Arg Gly His Asn Gly 65 70 75 80 Leu Leu Val Gln ValGlu Ile Ile Ile Asn Lys Val Thr Thr Leu Leu 85 90 95 Gln Asn Gly His IleLeu Val Gln Asn Lys Ser Val Ser Leu Pro Tyr 100 105 110 Asp His Thr TyrGln His Ile Phe Lys Tyr Gly Ile Tyr Thr Arg Leu 115 120 125 Arg Ser SerLeu Leu Pro Phe Thr Val Thr Trp His Asn Val His Gly 130 135 140 Gly IleAsn Ser Leu Trp Val Thr Leu Glu Ser Glu Leu Cys Thr Asp 145 150 155 160Met Cys Gly Leu Cys Gly Lys Gln Asn Val Ala Gly His Arg Asp Glu 165 170175 Leu Ile Arg Asp Ser Lys Leu His Asp His Arg Cys Lys Ile Arg Asp 180185 190 Pro Val Leu Arg Lys Asn His Ile Cys Arg Arg Phe Phe Leu Lys Thr195 200 205 Lys Asn Cys Leu Gln Asp Asn Asn Ser His Tyr His Arg Leu CysLys 210 215 220 Glu Asn Ile Cys Gly Phe Glu Asn Ser Gln Ser Ile Phe CysPro Phe 225 230 235 240 Phe Gln Glu Val Ala Ser Gln Cys Asn Gln Ser ArgIle Asn Arg Phe 245 250 255 Trp Arg Arg Leu Thr Lys Cys Ala Lys Pro ArgCys Pro Gly Asp Leu 260 265 270 Ile Tyr Glu Lys Lys Gly Pro Ala Phe IlePro Ser Cys Ser Asn Pro 275 280 285 Asn Pro Ala Pro Phe Tyr Gln Glu LeuThr Glu Thr Cys Ser Cys Pro 290 295 300 Glu Gly Lys Val Leu Asn Asn CysGlu Lys Gly Tyr Arg Cys Ile Pro 305 310 315 320 Lys Ser Ser Cys Ser CysGlu Phe Ala Gly Lys Thr Tyr Gly Asn Gly 325 330 335 Glu Ile Arg Ser SerArg Cys Gln Ser Cys Thr Cys Asp Gly Gly Lys 340 345 350 Trp Arg Cys SerGlu Asn Phe Cys His Arg Arg Cys Val Ile Glu Gly 355 360 365 Gln Phe ValThr Thr Phe Asp Gly Lys Gln Tyr Val Leu Pro Asn Lys 370 375 380 Cys LeuTyr Val Ala Ser Lys Gly Pro Asn Trp Ile Ile Ile Ile Glu 385 390 395 400Phe Ser Gln Lys Asn Leu Tyr Ile Arg Lys Val Thr Val Gln Leu Met 405 410415 Glu Glu Leu Phe Val Phe Lys Asn Asn Lys Val Leu Phe Asp Gly Gln 420425 430 Glu Ile Pro Glu Phe His Phe Ser Gly His Ala Gln Val Tyr Trp Val435 440 445 Ser Ser Met Phe Val Gln Val His Thr Thr Ile Gly Ile Asn PheGln 450 455 460 Ile Gln Met Ser Pro Glu Ile His Leu Phe Ile Asp Ala ProAsp Pro 465 470 475 480 Ser Ser Asp Lys Ile Lys Gly Leu Cys Gly Asn SerAsn Ser Asp Thr 485 490 495 Thr Asp Asp Phe Thr Thr Asn Ser Gly Ile IleGlu Asn Ser Ala Lys 500 505 510 Pro Phe Ala Met Ser Trp Ser Leu Leu AsnCys Phe Gly Asn Ile Pro 515 520 525 Thr Thr Cys Thr Asn Leu Glu Asn GluAsn Tyr Ala His Glu Lys Cys 530 535 540 Ala Val Leu Asn Gln Pro Thr GlyIle Phe Ala Lys Cys His Pro His 545 550 555 560 Ile Pro Thr Asp Tyr TyrTyr Thr Ala Cys Ile Gln Arg Ile Cys Asn 565 570 575 Ser Ala Gly Ser ArgArg Gln Gly Leu Cys Ile Gly Leu Ala Ser Tyr 580 585 590 Ala Lys Ala CysAla Gly Val Gly Val Val Ile Gly Asp Trp Leu Glu 595 600 605 His Gln GlyArg Pro 610 7 472 PRT Gasterosteus aculeatus 7 Met Thr Thr Gln Arg TrpIle Leu Ala Phe Cys Leu Ser Leu Ala Ser 1 5 10 15 Val Phe Gly Thr ValGlu Leu Phe Lys Thr Lys Glu Ile Gln Thr Tyr 20 25 30 Thr Cys Arg Thr PheGly Ser Gly Ile Val Gln Pro Phe Lys Gly Glu 35 40 45 Ser Tyr Tyr Val ArgSer Asp Cys Pro Phe Lys Leu Thr Ser Phe Asn 50 55 60 Val Asn Arg Gly GluTyr Ser Val Thr Ile Arg Arg Gly His Asn Gly 65 70 75 80 Leu Leu Val GlnVal Glu Ile Ile Ile Asn Lys Val Thr Thr Leu Leu 85 90 95 Gln Asn Gly HisIle Leu Val Gln Asn Lys Ser Val Ser Leu Pro Tyr 100 105 110 Asp His ThrTyr Gln His Ile Phe Lys Tyr Gly Ile Tyr Thr Arg Leu 115 120 125 Arg SerSer Leu Leu Pro Phe Thr Val Thr Trp His Asn Val His Gly 130 135 140 GlyIle Asn Ser Leu Trp Val Thr Leu Glu Ser Glu Leu Cys Thr Asp 145 150 155160 Met Cys Gly Leu Cys Gly Lys Gln Asn Val Ala Gly His Arg Asp Glu 165170 175 Leu Ile Arg Asp Ser Lys Leu His Asp His Arg Cys Lys Ile Arg Asp180 185 190 Pro Val Leu Arg Lys Asn His Ile Cys Arg Arg Phe Phe Leu LysThr 195 200 205 Lys Asn Cys Leu Gln Asp Asn Asn Ser His Tyr His Arg LeuCys Lys 210 215 220 Glu Asn Ile Cys Gly Phe Glu Asn Ser Gln Ser Ile PheCys Pro Phe 225 230 235 240 Phe Gln Glu Val Ala Ser Gln Cys Asn Gln SerArg Ile Asn Arg Phe 245 250 255 Trp Arg Arg Leu Thr Lys Cys Ala Lys ProArg Cys Pro Gly Asp Leu 260 265 270 Ile Tyr Glu Lys Lys Gly Pro Ala PheIle Pro Ser Cys Ser Asn Pro 275 280 285 Asn Pro Ala Pro Phe Tyr Gln GluLeu Thr Glu Thr Cys Ser Cys Pro 290 295 300 Glu Gly Lys Val Leu Asn AsnCys Glu Lys Gly Tyr Arg Cys Ile Pro 305 310 315 320 Lys Ser Ser Cys SerCys Glu Phe Ala Gly Lys Thr Tyr Gly Asn Gly 325 330 335 Glu Ile Arg SerSer Arg Cys Gln Ser Cys Thr Cys Asp Gly Gly Lys 340 345 350 Trp Arg CysSer Glu Asn Phe Cys His Arg Arg Cys Val Ile Glu Gly 355 360 365 Gln PheVal Thr Thr Phe Asp Gly Lys Gln Tyr Val Leu Pro Asn Lys 370 375 380 CysLeu Tyr Val Ala Ser Lys Gly Pro Asn Trp Ile Ile Ile Ile Glu 385 390 395400 Phe Ser Gln Lys Asn Leu Tyr Ile Arg Lys Val Thr Val Gln Leu Met 405410 415 Glu Glu Leu Phe Val Phe Lys Asn Asn Lys Val Leu Phe Asp Gly Gln420 425 430 Glu Ile Pro Glu Phe His Phe Ser Gly His Ala Gln Val Tyr TrpVal 435 440 445 Ser Ser Met Phe Val Gln Val His Thr Thr Ile Gly Ile AsnPhe Gln 450 455 460 Ile Gln Gly Gln His Arg Arg Pro 465 470 8 1419 DNAGasterosteus aculeatus 8 atgacaaccc agcgatggat tctggcattc tgcctttcactggcctcagt ttttggcaca 60 gtggaattgt tcaagaccaa agaaattcag acttacacttgcaggacatt tggcagcggg 120 atcgtccagc cttttaaggg ggagagttac tatgttcggtccgactgccc gttcaaactc 180 acaagcttca acgtcaaccg gggggaatat tctgtcaccatacggcgagg tcacaacggg 240 ctgttggtcc aagtcgagat catcatcaac aaagtcacaacacttttgca gaatggccac 300 atcctagtgc agaacaaaag tgtttcactt ccatacgaccacacctacca gcatatcttt 360 aaatacggca tctacactag actgaggagc tcgctgcttccttttactgt cacctggcac 420 aatgtacatg ggggaataaa ctctctgtgg gtgacactggagtcggagct gtgcaccgac 480 atgtgtggac tgtgtggaaa acaaaacgtt gcaggccacagggacgagtt gatcagagac 540 agcaagcttc atgaccacag atgtaaaatc agagatcctgtgttgcgaaa aaatcacata 600 tgccgtcgat tctttctgaa aaccaagaac tgtctgcaagacaacaattc tcactatcac 660 cgactctgta aagagaacat ttgtggcttt gaaaacagccagagcatctt ctgtcctttc 720 ttccaagaag ttgcaagcca gtgtaaccaa tcaagaatcaaccgattttg gagacgttta 780 accaaatgtg cgaagccgag gtgtccagga gacctgatttatgagaaaaa aggtccagca 840 tttattccca gctgctccaa cccgaaccct gcacccttctaccaggaact cactgaaacc 900 tgttcctgtc cagagggtaa agttttgaat aattgtgaaaagggctatcg ctgtatacct 960 aaatccagct gctcttgtga gtttgctggc aagacctacggaaacggaga aatacggagt 1020 tccaggtgtc agtcatgtac gtgtgatggt gggaaatggcgatgctcaga gaacttttgc 1080 cacagaagat gtgtcattga aggccagttt gtgacaacattcgatggaaa acaatatgtc 1140 ctccctaaca aatgtttata tgtggcttca aagggtcccaactggataat aataatagag 1200 ttttcgcaaa aaaacctcta cattagaaag gttacggttcagctcatgga ggaactgttt 1260 gtattcaaaa acaacaaggt tttgtttgat ggacaggagatccctgaatt ccatttttct 1320 ggtcatgctc aggtttactg ggtgtcctcc atgttcgttcaggtccacac gaccattggt 1380 ataaacttcc aaattcaggg gcagcatcgt cggccgtga1419 9 1457 DNA Gasterosteus aculeatus 9 catcaggcag catgacaacccagcgatgga ttctggcatt ctgcctttca ctggcctcag 60 tttttggcac agtggaattgttcaagacca aagaaattca gacttacact tgcaggacat 120 ttggcagcgg gatcgtccagccttttaagg gggagagtta ctatgttcgg tccgactgcc 180 cgttcaaact cacaagcttcaacgtcaacc ggggggaata ttctgtcacc atacggcgag 240 gtcacaacgg gctgttggtccaagtcgaga tcatcatcaa caaagtcaca acacttttgc 300 agaatggcca catcctagtgcagaacaaaa gtgtttcact tccatacgac cacacctacc 360 agcatatctt taaatacggcatctacacta gactgaggag ctcgctgctt ccttttactg 420 tcacctggca caatgtacatgggggaataa actctctgtg ggtgacactg gagtcggagc 480 tgtgcaccga catgtgtggactgtgtggaa aacaaaacgt tgcaggccac agggacgagt 540 tgatcagaga cagcaagcttcatgaccaca gatgtaaaat cagagatcct gtgttgcgaa 600 aaaatcacat atgccgtcgattctttctga aaaccaagaa ctgtctgcaa gacaacaatt 660 ctcactatca ccgactctgtaaagagaaca tttgtggctt tgaaaacagc cagagcatct 720 tctgtccttt cttccaagaagttgcaagcc agtgtaacca atcaagaatc aaccgatttt 780 ggagacgttt aaccaaatgtgcgaagccga ggtgtccagg agacctgatt tatgagaaaa 840 aaggtccagc atttattcccagctgctcca acccgaaccc tgcacccttc taccaggaac 900 tcactgaaac ctgttcctgtccagagggta aagttttgaa taattgtgaa aagggctatc 960 gctgtatacc taaatccagctgctcttgtg agtttgctgg caagacctac ggaaacggag 1020 aaatacggag ttccaggtgtcagtcatgta cgtgtgatgg tgggaaatgg cgatgctcag 1080 agaacttttg ccacagaagatgtgtcattg aaggccagtt tgtgacaaca ttcgatggaa 1140 aacaatatgt cctccctaacaaatgtttat atgtggcttc aaagggtccc aactggataa 1200 taataataga gttttcgcaaaaaaacctct acattagaaa ggttacggtt cagctcatgg 1260 aggaactgtt tgtattcaaaaacaacaagg ttttgtttga tggacaggag atccctgaat 1320 tccatttttc tggtcatgctcaggtttact gggtgtcctc catgttcgtt caggtccaca 1380 cgaccattgg tataaacttccaaattcagg ggcagcatcg tcggccgtga taacccagaa 1440 acaccgaaac tacaaca 1457

1. A substantially purified adhesive protein comprising the amino acidsequence of SEQ ID NO: 6 or fragments thereof.
 2. A substantiallypurified adhesive protein comprising the amino acid sequence of SEQ IDNO: 3 or fragments thereof.
 3. A substantially purified adhesive proteincomprising the amino acid sequence of SEQ ID NO: 7 or fragments thereof.4. A protein or protein fragment according to any one of claims 1-3,wherein said protein or fragment exhibits a repetitive structure ofCystein rich regions bisected by von Willebrand factor like proteinD-domains.
 5. An isolated and purified polynucleotide sequence encodinga protein of any one of claims 1-4.
 6. A polynucleotide sequence whichhybridises under stringent conditions to the sequence of claim
 5. 7. Anisolated and purified polynucleotide sequence comprising SEQ ID NO: 5 orfunctionally equivalent variants thereof, said sequence and variantscoding an adhesive protein.
 8. An isolated and purified polynucleotidesequence comprising SEQ ID NO: 2 or functionally equivalent variantsthereof, said sequence and variants coding an adhesive protein.
 9. Anisolated and purified polynucleotide sequence comprising SEQ ID NO: 8 orfunctionally equivalent variants thereof, said sequence and variantscoding an adhesive protein.
 10. An isolated and purified polynucleotidesequence comprising SEQ ID NO: 4 or functionally equivalent variantsthereof, said sequence and variants coding an adhesive protein.
 11. Anisolated and purified polynucleotide sequence comprising SEQ ID NO: 1 orfunctionally equivalent variants thereof, said sequence and variantscoding an adhesive protein.
 12. An isolated and purified polynucleotidesequence comprising SEQ ID NO: 9 or functionally equivalent variantsthereof, said sequence and variants coding an adhesive protein.
 13. Apolynucleotide sequence which is complementary to the sequence accordingto any one of claims 5-12.
 14. An expression vector comprising asequence according to any one of claims 5-12.
 15. A host cell containingthe vector of claim
 14. 16. A method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO: 6 or fragments thereof,the method comprising the steps of: culturing the host cell of claim 18under conditions suitable for the expression of the polypeptide; andrecovering the polypeptide from the host cell culture.
 17. A purifiedantibody binding specifically to a protein of any on of claim 1-4. 18.An antibody according to claim 17, characterized in that said antibodyis monoclonal.
 19. An antibody according to claim 17, characterized inthat said antibody is polyclonal.
 20. A method for the detection ofandrogenic compounds in biological systems, characterized in that saidmethod comprises the following steps: collection of samples of kidney orurinary bladder from sticklebacks, separation of proteins, transfer tomembrane, and detection of spiggin using antibodies directed to theprotein of claim 1, 2, 3 or
 4. 21. A method for the detection ofandrogenic compounds in biological systems, characterized in that saidmethod comprises the following steps: collection of samples of kidney orurinary bladder from sticklebacks, coating of ELISA plates with samplesand standard, blocking and washing wells, addition of a monoclonalantibody directed to the protein of claim 1, 2, 3 or 4, incubation andwashing, addition of secondary antibody, incubation and washing, anddetection of signal.
 22. A method for the detection of androgeniccompounds in biological systems, characterized in that said methodcomprises the following steps: collection of samples of kidney orurinary bladder from sticklebacks, extraction of RNA, separation of RNAon agarose gel, transfer to nylon membrane, prehybridisation followed byhybridisation with labelled spiggin cDNA or cRNA, incubation andwashing, and detection of specific signal.
 23. A kit for the detectionof androgenic compounds, characterized in that said kit comprises anantibody according to any one of claims 17-19.
 24. A kit for thedetection of androgenic compounds, characterized in that said kitcomprises and antibody according to any one of claims 17-19 and aprotein standard based on the protein of claim 1, 2, 3 or 4, buffers,secondary antibodies, detection substrate, specification chart for theprimary and secondary antibodies and the peptide standard andinstructions for use.
 25. A therapeutic composition, characterized inthat it comprises an effective amount of a protein according to any oneof claims 1-4.
 26. A therapeutic composition according to claim 25,characterized in that it further comprises a compound selected amongantibacterial, antiviral, antifungal, analgesic, moisturising, anddehydrating compounds.
 27. A therapeutic composition according to claim25, characterized in that it further comprises an antibiotic compound.28. A wound dressing, characterized in that it comprises an effectiveamount of a protein according to any one of claims 1-4.
 29. An implant,characterized in that it comprises a protein according to any one ofclaims 1-4.
 30. Use of a polypeptide according to any one of claims 1-4,for the manufacture of a product for use in or on the body of a mammal.31. Use of a polypeptide according to any one of claims 1-4, for themanufacture of a medicament.
 32. Use according to claim 30,characterized in that the product for use in or on the body of a mammalis chosen among the following products: stents, sutures, wound closurestaples, surgical tape, surgical glue, hemostatic fillers andcompresses, protective wound covers and/or coatings,
 33. An adhesive forapplication in moist environments, characterized in that it comprises aneffective amount of a protein according to any one of claims 1-4produced in a transgenic host cell.
 34. A sealant for application inmoist environments, characterized in that it comprises an effectiveamount of a protein according to any one of claims 1-4 produced in atransgenic host cell.